Creatine kinase (EC 2.7.3.2) is essential for maintaining proper intracellular ATP:ADP ratios and phosphocreatine pool size and plays a prominent role in cardiac and skeletal muscle energy metabolism. The creatine kinase genes comprise a small gene family that is differentially regulated during myogenesis and in tissues. Creatine kinase undergoes a developmental isoenzyme switch in heart and skeletal muscle characterized by replacement of the BB isoenzyme by the mature MM isoenzyme. A number of pathologic and physiologic stimuli modulate the expression of the creatine kinase genes and are associated with partial reversion to the fetal isoenzyme composition. Elucidation of factors modulating expression of the creatine kinase genes should provide insights relevant to the nature of control of expression of diverse myocardial proteins in response to pathologic and physiologic stimuli of potential importance to the understanding of disease states that affect the heart. We will complete the characterization of the human M creatine kinase gene enhancer critical for the tissue-specific and developmental regulation of the M creatine kinase gene in transfected C(2)C(12) myoblasts undergoing differentiation in culture. We will determine whether the human B creatine kinase gene is regulated at the level of transcription and/or by post-transcriptional mechanisms in C(2)C(12) myoblasts undergoing differentiation and characterize the cis-acting sequence elements which confer regulation during development by transfecting C(2)C(12) cells in culture with chimeric plasmids containing regulatory sequences of the human B creatine kinase gene fused with the bacterial chloramphenicol acetyltransferase (CAT) gene in a transient expression system. We will define the cis-acting sequence elements present in the human M and B creatine kinase genes which confer regulation of expression in heart by transfecting cardiac myocytes harvested from neonatal rat hearts with chimeric plasmids in a transient expression system. Ultimately, with the use of sequence-specific DNA affinity chromatography and by screening cDNA expression libraries with cis-acting DNA sequence elements we will isolate and characterize the trans-acting factors and clone the cDNAs, and genes encoding these proteins which interact with the cis-acting elements present in the human creatine kinase genes and regulate their expression.